Project Summary Doxorubicin (DOX; Adriamycin) is an antitumor antibiotic widely used drug for the treatment of a variety of cancers. Unfortunately, the clinical use of this drug is limited due to severe, dose-dependent cardiac side effects that lead to irreversible chronic cardiomyopathy followed by congestive heart failure. Novel therapeutic approaches using cell transplantation are being studied to regenerate infarcted and DOX-induced cardiomyopathy (DIC) myocardium. However, there is no data yet available describing the ability of embryonic stem (ES) cells or factors released from ES cells to be used in DIC. Using a cell culture model system, our preliminary data suggest that factors released from ES cells contain anti-apoptotic, anti-fibrotic and growth factors (insulin and hepatocyte growth factors, IGF-1 and HGF) necessary inhibit adverse remodeling (including reduced apoptosis, fibrosis, cytoplasmic vacuolization and myofibrillar loss) and to stimulate resident cardiac stem cells (CSCs). We also demonstrate that TGF2 enhances ES cells derived embryoid body (EB) proliferation and cardiac myocyte differentiation. Overall, we propose central hypothesis that TGF2 treated cardiac committed (TCC)-ES cells and factors released from ES cells in DIC following transplantation will demonstrate engraftment, differentiation, remodeling, cardiac regeneration, and ultimately, function. Furthermore, our preliminary data also demonstrate the differentiation of c-kit+CSCs in DIC following ES-CM or TCC-ES cells transplantation. So, we also hypothesize that factors released (IGF-1 and HGF) from transplanted ES-CM or TCC-ES cells stimulates c-kit+CSCs and enhance cardiac regeneration. Accordingly the specific aims are as follow: Aim 1: a) Determine if intraperitoneal injection of (a) ES-CM attenuates DIC. (b) TCC-ES cells attenuate apoptosis and enhance cardiac repair and regeneration in DIC. Aim 2: Determine if intramyocardial injection of TCC-ES cells or ES-CM enhance cardiac repair and regeneration in DIC. Aim 3: Determine if transplantation of factors released from ES cells enhance c-kit+ CSCs proliferation, differentiation and regeneration in DIC. Aim 4: Determine the mechanisms of inhibited apoptosis and fibrosis following TCC- ES cell or ES-CM transplantation in DIC. The results of the proposed studies should help to define the impact of TCC-ES cells or ES-CM on cardiac repair and regeneration in DIC. We will also define mechanisms of CSCs proliferation and differentiation, and remodeling that may play a role in improved cardiac function in DIC following transplantation. Moreover, the use of TCC-ES cells or ES-CM that release various factors that stimulate tissue repair and regeneration may have promise as a new therapeutic strategy in the field of regenerative medicine.